Modified pectins, compositions and methods related thereto

ABSTRACT

The present invention provides compositions of modified pectin and for preparing and using them.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional App. No.60/556,674 filed Mar. 26, 2004, entitled “MODIFIED PECTINS, COMPOSITIONSAND METHODS RELATED THERETO,” the disclosure of which is incorporated byreference in its entirety.

FIELD OF THE INVENTION

The invention relates in general to the fields of polysaccharidechemistry and oligosaccharide chemistry, and more particularly to thechemistry of naturally-occurring pectins and methods for making andusing modified pectins.

BACKGROUND OF THE INVENTION

Modified pectins have long been recognized as being useful insuppressing the metastasis of cancer cells. This effect is thought to bedue to binding of the modified pectin to galectins, in particulargalectin-3, and possibly to other as-yet unidentified saccharide-bindingcell surface receptors. Several compositions of modified pectins havebeen described. See Platt et al. “Modulation of Lung Colonization ofB16-F1 Melanoma Cells by Citrus Pectin” J. Natl. Cancer Inst.84(6):438-442(1992); Inohara et al. “Effects of Natural ComplexCarbohydrate (citrus pectin) on Murine Melanoma Cell Properties Relatedto Galectin-3 function” Glycoconjugate J. 11:527-532(1994); Pienta etal. “Inhibition of spontaneous metastasis in a Rat Prostate Cancer Modelby Oral Administration of Modified Citrus Pectin” J. Natl. Cancer Inst.87:348-353(1995); U.S. patent application Ser. Nos. 08/024,487,08/819,356, 2003/0013682, 2003/0004132, and 2002/0107222, and U.S. Pat.Nos. 6,423,314, 5,681,923, 5,834,442, and 5,895,784. The disclosures ofeach of these applications and patents are incorporated herein byreference in their entirety. Synthetic carbohydrate derivatives havealso been reported to bind to and block galectin-3. See PCT applicationWO 02/057284.

Examples of modified pectins of the type described previously aredescribed by formulas I-IV below:—[α-D-GalpA-(1→4)-α-D-GalpA] _(n)—  (I)

In the above representations, n is an integer greater than 1, X_(n-1)represents a short side-chain of neutral sugar residues, Galp isgalactose, Rhap is rhamnose, GalpA is galacturonic acid and Araf isarabinose. X can be any of several sugars found in pectin side chains,including but not limited to β-Apif, β-Rhap, α-Fucp, β-GlcpA, α-GalpA,β-GalpA, β-DhapA, Kdop, β-Acef, α-Galp, and α-Arap.

The existing methods used to prepare modified pectins generally sufferfrom poorly controlled chemical processes and difficult productisolation and purification processes. These factors either separately orcombined typically result in widely varying therapeutic activities,molecular weights, polydispersities, concentrations, monosaccharidecompositions, linkage makeup, potency and impurity profiles. Many ofthese processes employ organic solvents, which can create explosionhazards and toxicological effects from residual solvent and requireexpensive disposal of solvent waste.

SUMMARY OF THE INVENTION

The invention provides modified pectin compositions and methods ofproducing and using them. The modified pectin compositions describedherein may have improved potency, purity and composition uniformity, andthe methods of manufacture permit these benefits to be achieved reliablyand reproducibly. In certain embodiments, the modified pectincompositions are substantially free of ethanol and acetone (e.g., haveless than 1% of either or both solvents).

In certain embodiments, the modified pectin consists essentially of abackbone comprising homogalacturonan and/or rhamnogalacturonan I, havingneutral sugar side chains and a low degree of neutral sugar branchingdependent from the backbone. In certain embodiments, the modified pectinis de-esterified and partially depolymerized, so as to have a disruptedrhamnogalacturonan backbone. The compositions are preferably aqueoussolutions containing at least 0.5%, 1%, 5% or 10% by weight of themodified pectin, e.g., up to about 10% or 15% by weight. In anotherembodiment, the modified pectin forms a colloidal solution in water. Thesize of the colloidal particles may be less than 1 μm in diameter,preferably less than 0.65 μm, and most preferably less than 0.2 μm.

The invention also provides methods for manufacturing modified pectin,comprising one or more of the following steps: partially depolymerizinga pectin polymer by disrupting the rhamnogalacturonan/homogalacturonanbackbone, de-esterifying galacturonic acid moieties in the backbone,breaking down side chains of neutral sugars, and optionally creatingand/or isolating a colloidal suspension of the resulting product inwater. In certain embodiments, the process of the invention provides asubstantially ethanol- and acetone-free product suitable for parenteralor oral administration.

The present invention relates in part to a modified pectin material thatinhibits cancer cell proliferation with an IC₅₀ less than 100 μg/mL,less than 75 μg/mL, less than 50 μg/mL, e.g., with an IC₅₀ in the rangeof 25-100 μg/mL, 25-75 μg/mL, or 30-50 μg/mL, or even less than 1 μg/mL,e.g., with an IC₅₀ in the range of nanograms/mL.

The present invention further relates to a composition comprising amodified pectin material, such as a de-esterified and partiallydepolymerized modified pectin, substantially free of modified pectinshaving molecular weights below 25 kD.

The present invention further relates to a modified pectin made bypassing modified or unmodified (commercial) pectin through a tangentialflow filter, e.g., a filter having a pore size below 1, 0.65, 0.45, or0.22 μm, such as a 0.2 μm filter. The pectin may be filtered as anaqueous solution (e.g., at a concentration of 1 to 20 mg/mL, preferably3-8 mg/mL, most preferably 4-6 mg/mL) comprising 0-25%, preferably10-20%, w/w ethanol. The solution may have a pH in the range of 2.5 to10, preferably 3.0-7.5, most preferably 5.0 to 7.0.

In certain embodiments, the present invention provides a method forproducing a modified pectin comprising partially depolymerizing a pectinpolymer by disrupting homogalacturonan and/or rhamnogalacturonanbackbones of the pectin polymer, de-esterifying galacturonic acidmoieties in the backbone, breaking down side chains of neutral sugarsinto low molecular weight sugars, and collecting material remainingafter ultrafiltration using a filter having a nominal 30 kD cutoff.

A modified pectin material as described herein preferably has an averagemolecular weight from 50-200 kD, 70-175 kD, 70-150 kD, 80-150 kD, oreven 80-100 kD as measured by Gel Permeation Chromatography (GPC) withMulti Angle Laser Light Scattering (MALLS) detection.

A modified pectin as described or produced herein may consistessentially of a homogalacturonan backbone with small amounts ofrhamnogalacturonan therein, wherein the backbone has neutral sugar sidechains having a low degree of branching dependent from the backbone. Incertain embodiments, the galacturonic acid subunits of the backbone arepartially de-esterified, and in particular embodiments, the galacturonicacid subunits of the backbone are substantially de-esterified.

The compositions as described or produced herein may be formulated aspharmaceutical composition further comprising a pharmaceuticallyacceptable excipient. Such compositions may be aqueous solutions ofmodified pectin at a concentration of at least 0.5, 1, 5, or even 10mg/mL, e.g., 0.5-10, 1-10, 5-10 mg/mL. Other forms include oral dosageforms, topical dosage forms, and inhalable formulations, such asinhalers comprising a modified pectin as described herein.

In another aspect, the present invention provides a process formanufacturing a de-esterified and partially depolymerized modifiedpectin, comprising providing a slurry of pectin in a water-miscibleorganic solvent (such as ethanol), combining the slurry with water todissolve the pectin and form a solution, and treating the resultingsolution with acid, base, or both to break down the pectin.

In yet another aspect, the present invention provides a process formanufacturing a de-esterified and partially depolymerized modifiedpectin, comprising treating a solution of pectin with acid, base, orboth to break down the pectin, neutralizing the solution, and purifyinga solution of the modified pectin by ultrafiltration (such as bytangential flow filtration).

In another aspect, the present invention provides a process formanufacturing a de-esterified and partially depolymerized modifiedpectin, comprising maintaining a solution at an alkaline pH between 9and 12 (e.g., from 10 to 11) for up to 4 hours, lowering the pH of thesolution to an acidic pH between 2 and 5 (e.g., from 2.5 to 3.5) for upto several days, and neutralizing the solution.

The present invention further relates to a method of producing modifiedpectin by passing modified (e.g., physically, chemically, and/orbiologically-modified pectin as described herein) or unmodified(commercial) pectin through a tangential flow filter, e.g., a filterhaving a pore size below 1.0, 0.65, 0.45, or 0.22 μm, such as a 0.2 μmfilter. The pectin may be filtered as an aqueous solution (e.g., at aconcentration of 1 to 20 mg/mL, preferably 3-8 mg/mL, most preferably4-6 mg/mL) comprising 0-25%, preferably 10-20%, w/w ethanol. Thesolution may have a pH in the range of 2.5 to 10, preferably 3.0-7.5,most preferably 5.0 to 7.0.

A process for producing modified pectin as described herein may furthercomprise precipitating modified pectin from the solution and washing themodified pectin with ethanol after neutralizing the solution and beforepurifying a solution of the modified pectin. Additional steps mayinclude one or more of: adjusting the solution to iso-osmolality,clarifying the solution, subjecting a solution of the modified pectin tomicrofiltration, and lyophilizing the modified pectin. This process mayfurther comprise treating the solution to reduce the concentration ofendotoxins, and/or purifying a solution of the modified pectin byultrafiltration to remove low molecular weight material.

The invention further relates to a modified pectin, such asde-esterified and partially depolymerized modified pectin produced byany method described herein, and pharmaceutical compositions thereof.

The present invention also provides pharmaceutical packages. In one suchembodiment, a pharmaceutical package comprises a vial or ampoulecontaining a pharmaceutical composition comprising a modified pectin asdescribed herein and a pharmaceutically acceptable excipient as anaqueous solution suitable for injection, and instructions foradministering the composition to a patient in need thereof. In anotherembodiment, a pharmaceutical package comprises a plastic bag containingfrom 100 ml to 2 L of a pharmaceutical composition as described hereinas a solution suitable for intravenous administration, and instructionsfor administering the composition to a patient in need thereof. In yetanother embodiment, a pharmaceutical package comprises a solution ofmodified pectin as described herein and instructions for diluting thesolution of modified pectin to a concentration suitable foradministration to a patient intravenously or by injection. In still yetanother embodiment, a pharmaceutical package comprises a pharmaceuticalcomposition as described herein and instructions for diluting thecomposition to a concentration suitable for administration to a patientintravenously or by injection.

In still yet another aspect, the invention provides a method ofinhibiting a cell proliferation process in a patient by administering amodified pectin material as described herein to a patient, therebyinhibiting cell proliferation in the patient. In certain embodiments,the cell proliferation process is angiogenesis or cancer, e.g., selectedfrom renal cell cancer, Kaposi's sarcoma, chronic leukemia, chroniclymphocytic leukemia, breast cancer, sarcoma, myeloma, ovariancarcinoma, rectal cancer, throat cancer, melanoma, lymphoma,mesothelioma, colon cancer, bladder cancer, mastocytoma, lung cancer,liver cancer, mammary adenocarcinoma, pharyngeal squamous cellcarcinoma, prostate cancer, pancreatic cancer, gastrointestinal cancer,and stomach cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the effect of microfiltration pore size ontherapeutic effectiveness.

FIG. 2 demonstrates the effect of microfiltration feed pH on therapeuticeffectiveness.

FIG. 3 demonstrates the effect of microfiltration feed ethanol contenton therapeutic effectiveness.

FIG. 4 demonstrates the effect of modified pectin prepared by prior andnew methods on DNA synthesis inhibition.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

The present invention discloses methods for the production of modifiedpectins with therapeutic effects, e.g., against cancer cells. Asdescribed herein, pectin can be modified by chemical, physical and/orbiological, including enzymatic, means.

The invention also describes compositions composed in part or entirelyof modified pectin that are suitable for the treatment of cancer. By wayof example, useful compositions of modified pectin include dry powders,suspensions, gels or aqueous solutions. These compositions may consistessentially of modified pectin without any excipients, or they may be incombination with one or more pharmaceutically acceptable excipients.

A modified pectin composition of the present invention comprises orconsists essentially of a homogalacturonan backbone with small amountsof rhamnogalacturonan I interspersed therein, with neutral sugar sidechains, and has a low degree of neutral sugar branching dependent fromthe backbone. In certain embodiments, the modified pectin isde-esterified and partially depolymerized, so as to have a disruptedhomogalacturonan backbone.

Modified pectin can be administered by any of a variety of routes.Examples of such delivery methods include oral suspensions, gels, andtablets, injectable solutions (intravenous, intraperitoneal), inhalablepowders, and suspensions. Preferred compositions are aqueous solutions,oral tablets, and gels.

As used herein, the term “modified pectin” herein refers to any pectinthat has been structurally modified, e.g., by chemical, physical, orbiological (including enzymatic) means, or by some combination thereof.Non-limiting examples of such modification to the pectin structureinclude (but are not limited to) de-esterification, hydrolysis,oxidation and/or reduction of sugar moieties, functionalization of sugarmoieties, conformational changes, and changes in molecular weight,linkage, and states of aggregation. In preferred embodiments, thestructural modification includes one or more of de-esterification andhydrolysis. In other preferred embodiments, the structural modificationincludes reduction in particle size and/or aggregation states.

Modified pectin may be produced by chemical means, e.g., any chemicalreaction or process that disrupts or changes chemical bonds of thepectin structure, such as covalent or ionic bonds. By way of exampleonly, chemical bonding may be disrupted or formed by catalysis,hydrolysis, substitution, elimination, reduction, oxidation, and radicalreactions. In certain embodiments, modified pectin according to theinvention is produced by a process that includes hydrolysis, which ispreferably catalyzed, e.g., by an acidic or basic reagent or both.

Pectin may also be modified by physical means. Physical means are meantto include non-chemical or -biological means that alter the structure ofpectin. Such physical means include, but are not limited to heat, cold,freeze/thaw, irradiation, shear, ultra-high shear, use of cosolvents,and filtration.

Pectin may also be modified by biological means. Biological means aremeant to include by way of example, enzymatic degradation of pectin.Finally, the invention contemplates the preparation of modified pectinby synthetic and/or recombinant methods. Like the production ofrecombinant proteins, complex polysaccharides might be engineered via aseries of synthetic reactions or by recombinant methods.

In certain embodiments, pectin may be modified by a combination of theforegoing methods, such as chemical modification followed by physical orbiological modification.

Typically, processes for making modified pectin result in a mixture ofmodified pectin and a number of impurities such as low molecular weightby-products, salts, co-solvents and inactive modified pectin. In certainembodiments of the invention, these impurities are acceptable and arenot removed from the modified pectin. In other embodiments, theimpurities are either reduced or removed entirely from the modifiedpectin composition. According to the invention, impurities can beremoved by methods known in the art. By way of example, these methodsinclude filtration, microfiltration, ultrafiltration, chromatography,centrifugation, extraction, drying, precipitation and dialysis. Incertain preferred embodiments, the purification includesultrafiltration.

Certain previously described processes of making modified pectin resultin the formation of a modified pectin with a polydisperse molecularweight and particle size and low or varying degrees of therapeuticactivity against cancer cell lines. In one embodiment of the invention,the polydispersity of the modified pectin is reduced to less than 5,preferably less than 4 or 3 and most preferably less than 2.5 toincrease the modified pectin's effectiveness against cancer cell linesand, in turn, cancer. In preferred embodiments, the particle sizepolydispersity of the modified pectin is modified by microfiltration. Inother preferred embodiments, the molecular weight polydispersity of themodified pectin is modified by ultrafiltration. In a certainembodiments, the modified pectin has an average particle size of lessthan 1 μm, preferably less than 0.65 μm and even more preferably lessthan 0.2 μm as measured by dynamic light scattering or atomic forcemicroscopy.

In one embodiment, the method comprises the following acts or any subsetthereof:

dissolving the pectin in water, e.g., at a concentration of 0.1 to 40mg/mL, preferably 1 to 20 mg/mL, more preferably 5 to 15 mg/mL and mostpreferably about 10 mg/mL;

maintaining an alkaline (basic) pH, e.g., in a range of 7 to 13,preferably 9 to 12, more preferably 10 to 11, and most preferably about10.3 to 11, for a duration of about 24 hours, preferably less than 12hours, more preferably less than 1 hour and most preferably about 10-30minutes;

maintaining an acidic pH, e.g., a pH in a range of 1 to 6.9, preferably2 to 5, more preferably 2.5 to 3.5, and most preferably about 3, for aduration of about 24 hours, preferably less than 12 hours, morepreferably less than 1 hour and most preferably about 5-15 minutes;

neutralizing the solution to a pH between 4 and 8, preferably thesolution is neutralized to a pH between 5 and 8, and more preferably toa pH between 6 and 8, and most preferably to a pH between 6 and 7;

precipitating and washing the modified pectin with ethanol;

dissolving the washed precipitate in water;

filtering the solution, preferably by microfiltration, e.g., through a0.2 μm filter, such that the modified pectin passes through the filter;and

concentrating and diafiltering the solution of modified pectin usingultrafiltration (e.g., using a 30 kD membrane).

In various embodiments, the processes outlined include sterilefiltration, sterile filling, removal of endotoxins, lyophilization, or acombination of any of these.

The present invention contemplates several different types of modifiedpectin compositions with different attributes and potential uses. In onecomposition of the invention, the modified pectin is in a solutionhaving a modified pectin concentration of at least 0.1 mg/mL, preferablyat least 1 mg/mL, more preferably at least 5 mg/mL, at least 7 mg/mL, atleast 10 mg/mL, or even at least 15 mg/mL. In certain embodiments, thesolution is a 10-30% aqueous ethanol solution. In certain otherembodiments, the modified pectin solution is substantially free ofethanol and acetone. In yet another preferred embodiment, the modifiedpectin composition described above has no ethanol or other organicsolvent, such as acetone, ether, dimethylsulfoxide, ethyl acetate, etc.

In one embodiment, the invention provides a modified pectin comprisingrhamnogalacturonan and/or homogalacturonan backbone with neutral sugarside chains, and having a low degree of neutral sugar branchingdependent from the backbone. In certain embodiments, the modified pectinis deesterified and partially depolymerized, so as to have a disruptedrhanmogalacturonan backbone.

In one embodiment, the modified pectin includes a copolymer ofgalacturonic acid and rhamnogalacturonan I in which at least some of thegalactose- and arabinose-containing sidechains are still attached. Inpreferred embodiments, the modified pectin has an average molecularweight of 50-200 kD, preferably 70-200 kD, more preferably 70-150 kD asmeasured by Gel Permeation Chromatography (GPC) with Multi Angle LaserLight Scattering (MALLS) detection.

In certain embodiments, the compositions are suitable for parenteraladministration to a mammal, most preferably by injection or intravenousinfusion. The composition may be adapted for direct injection orintravenous infusion, or for addition to an intravenous drip solutionfor gradual infusion, through appropriate use of excipients andpackaging and delivery means well known in the art. In certainembodiments the compositions comprise one or more pharmaceuticallyacceptable excipients, such as water, pharmaceutically acceptablebuffers, stabilizers, local anesthetics, and the like, as described ingreater detail below.

In another aspect, the invention provides a pharmaceutical package,comprising a vial or ampoule containing a modified pectin according tothe invention in the form of a reconstitutable powder or a solutionsuitable for injection or infusion, e.g., together with instructions foradministering the composition to a patient in need thereof. Instructionsinclude but are not limited to written and/or pictorial descriptions of:the active ingredient, directions for diluting the composition to aconcentration suitable for administration, suitable indications,suitable dosage regimens, contraindications, drug interactions, and anyadverse side-effects noted in the course of clinical trials.

The invention also provides a method for conducting a medical assistancereimbursement program, the method comprising: (a) providing areimbursement program that permits, for prescription of a composition,solution, or depolymerized pectin of the invention for treating acancer, at least partial reimbursement to a healthcare provider orpatient, or payment to a drug distributor; (b) processing one or moreclaims for reimbursement of the cost of a prescription of thecomposition, solution, or depolymerized pectin for treating a cancer;and (c) reimbursing the healthcare provider or patient, or paying a drugdistributor, at least a portion of the cost of said prescription.

In certain embodiments, a solution of modified pectin as described aboveis lyophilized, e.g., by methods well-known in the art, to provide a drysolid formulation adapted for reconstitution into a solution suitablefor intravenous administration. Such lyophilized compositions mayoptionally comprise additives, such as wetting agents and sugars, topromote dissolution.

In certain embodiments, the invention provides a process for dissolvingdry pectin in water at the beginning of the modification process. Theprocess of dissolving the pectin in water is not straightforward, due tothe unusual properties of dry hydrocolloid powders. Upon contact withwater, the pectin particles rapidly swell and become sticky, and theytend to crowd against and adhere to any neighboring particles. Theresult is a sticky mass that dissolves very slowly, due to the limitedsurface area in contact with the water. It is preferable to keep theparticles separated from one another long enough for them to disperseinto the water, where the individual particles remain separated and candissolve much more quickly.

Methods for separating pectin particles at the time of their dispersionin water include, but are not limited to, (a) use of an eductor funnel,(b) addition of a sugar, (c) suspension in a non-solvent, (d) use ofhigh shear, and (e) slow, controlled addition of reagents. In a powdereductor funnel, the pectin particles are separated by a stream of airjust before they contact the water. When a dry blend of several partssugar to one part pectin is dispersed into water, the sugar particlesseparate the pectin particles, allowing the pectin particles to hydrateand expand without contacting their neighbors. Suspension in anon-solvent, such as vegetable oil, glycerin, ethanol, or corn syrup,results in the pectin particles becoming wetted and separated from oneanother without swelling or becoming sticky. The suspension is then beadded to water with agitation. With the use of high shear,rapidly-moving water separates adhering particles and breaks up anylumps, maintaining a high surface area and enabling quick hydration. Inthe case of slow controlled addition, pectin may simply be added at aslow controlled rate to a stirring solution of water. Preferably, therate of addition is sufficiently slow and the mixing speed issufficiently great to allow the pectin to be rapidly dispersed andhydrated. Controlled pectin addition can be accomplished manually orwith the use of a powder addition device, such as an automated solidsmetering device outfitted with an adjustable rate extruding screw.

In certain embodiments, modified pectins of the invention are describedby either or both of formulas I and II below, and it is to be understoodthat variants of these general formulae may be prepared and utilized inaccord with the principles of the present invention.

Homogalacturonan—[α-GalpA-(1→4)-α-GalpA] _(n)—  (I)

In the formulae above, m is ≧0, n, o and p are ≧1, X is α-Rhap; andY_(m) represents a linear or branched chain of sugars (each Y in thechain Y_(m) can independently represent a different sugar within thechain). The sugar Y may be, but is not limited to, any of the following:α-Galp, β-Galp, β-Apif, β-Rhap, α-Rhap, α-Fucp, β-GlcpA, α-GalpA,β-GalpA, β-DhapA, Kdop, β-Acef, α-Araf, β-Araf, and α-Xylp.

It will be understood that natural pectin does not possess a strictlyregular repeating structure, and that additional random variations arelikely to be introduced by partial hydrolysis of the pectin, so that theidentity of Y_(m) and the values of n and o may vary from one iterationto the next of the p repeating units represented by formula II above.

Abbreviated sugar monomer names used herein are defined as follows:GalA: galacturonic acid; Rha: rhamnose; Gal: galactose; Api:erythro-apiose; Fuc: fucose; GlcA: glucuronic acid; DhaA:3-deoxy-D-lyxo-heptulosaric acid; Kdo: 3-deoxy-D-manno-2-octulosonicacid; Ace: aceric acid (3-C-carboxy-5-deoxy-L-lyxose); Ara: arabinose.Italicized p indicates the pyranose form, and italicized f indicates afuranose ring.

II. Definitions

The term “healthcare providers” refers to individuals or organizationsthat provide healthcare services to a person, community, etc. Examplesof “healthcare providers” include but are not limited to doctors,hospitals, continuing care retirement communities, skilled nursingfacilities, sub-acute care facilities, clinics, multispecialty clinics,freestanding ambulatory centers, home health agencies, and HMO's.

A “patient” or “subject” to be treated by a method of the invention canmean either a human or non-human subject.

The term “IC₅₀” means the concentration of an agent that produces a 50%reduction in the effect compared to when there is a complete absence ofthe agent being tested for IC₅₀.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable excipient” as used herein meansa pharmaceutically acceptable material, composition or vehicle, such asa liquid or solid filler, diluent, lubricant, binder, carrier,humectant, disintegrant, solvent or encapsulating material, that oneskilled in the art would consider suitable for rendering apharmaceutical formulation suitable for administration to a subject.Each excipient must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation, as well as“pharmaceutically acceptable” as defined above. Examples of materialswhich can serve as pharmaceutically acceptable excipients include butare not limited to: sugars, such as lactose, glucose and sucrose;starches, such as corn starch and potato starch; cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; silica,waxes; oils, such as corn oil and sesame oil; glycols, such as propyleneglycol and glycerin; polyols, such as sorbitol, mannitol andpolyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents; alginic acid; pyrogen-free water; isotonicsaline; Ringer's solution; and other non-toxic compatible substancesroutinely employed in pharmaceutical formulations.

By “substantially free” of ethanol or another solvent, it is meant thatthe compositions of the invention contain less than 5% of that solventby weight. In preferred embodiments, the subject compositions containless than 2%, less than 1%, and more preferably less than 0.5% ethanolby weight, and preferably less than 1% or 0.5% acetone by weight.

By “substantially free” of modified pectins having a certain molecularweight below a certain number, it is meant that the composition has lessthan 1%, preferably less than 0.5% or even less than 0.1%, of modifiedpectins having a molecular weight below that number.

A “therapeutically effective amount” of a compound, such as a modifiedpectin of the present invention, with respect to the subject method oftreatment, refers to an amount of the compound(s) in a preparationwhich, when administered as part of a desired dosage regimen to asubject, slows or arrests the progress of the disease or conditionsought to be treated.

III. Methods for Preparing Modified Pectins

A. Choice of Pectin Starting Material

Pectin is a major constituent of plant cell walls, and is a combinationof at least three principal pectic polysaccharides, which are believedto be covalently linked within the cell wall: homogalacturonan (HG),rhamnogalacturonan I (RG-I), and rhamnogalacturonan II (RG-II).

HG is a linear homopolymer of 1,4-linked α-D-galacturonic acid, methylesterified to varying degrees at C-6. Depending on the species of plant,the backbone galacturonic acid units may be C-3 substituted withO-acetyl residues.

RG-I is a heterologous group of polysaccharides that contain a backboneof the repeating disaccharide [→4)-α-D-GalpA-(1→2)-α-L-Rhap-(1→].Between 20 and 80% of the Rhap residues are substituted at C-4 withneutral oligosaccharide side chains containing linear and branchedα-L-Araf and β-D-Galp residues. The backbone GalpA residues of RG-I arenot typically substituted with polysaccharides, although they may beO-acetylated at C2 or C3.

RG-II has a more highly conserved structure, with a backbone usuallycomposed of at least seven to nine 1,4-linked α-D-GalpA residues, towhich four complex oligosaccharide side chains are typically attached atC-2 and/or C-3.

Pectin itself is thought to be a heteropolysaccharide with a backbonecomposed of alternating HG (“smooth regions”) and RG (“hairy regions”).The smooth regions are linear polymers of 1,4-linked α-D-galacturonicacid.

The highly branched “hairy regions” feature neutral sugar units(typically D-galactose or L-arabinose or xylose attached by glycosidiclinkages to the C4 atoms of the rhamnose units, and/or to the C2 or C3atoms of the galacturonic acid units. Depending upon the extractionprocess used, the hairy regions are partially or largely degraded duringthe manufacture of commercial pectin, leaving intact the smoothpolygalacturonic acid regions, with a smaller number of neutral sugarunits still attached to or embedded in the main linear chain. The methylgalacturonate ester groups survive the extraction process, althoughdegree of methyl esterification may be reduced in subsequent processingsteps to provide commercial pectins having various utilities.

The degree of methyl esterification in most commercial pectins variesfrom 0-90%. If 50% or more of the carboxyl groups are esterified thepectin is referred to as a “high ester” or “high methoxyl” pectin”. Ifless than 50% of the carboxyl groups are esterified then the pectin isreferred to as a “low ester” or “low methoxyl” pectin. Pectin having fewor no esterified groups is referred to as pectic acid.

The choice of the starting pectin material affects the characteristicsof the final product. However, in choosing a starting pectin, importantthings to consider are molecular weight, degree of esterification,monosaccharide content, linkage, polydispersity and so forth. In apreferred embodiment, the starting pectin contains roughly 50-60 mole %esterification or higher, has a molecular weight of greater than 150 kD,and has a particular monosaccharide content, e.g., galactose content,greater than or equal to 5%. In one embodiment of the invention thestarting pectin composition may comprise approximately equal amounts ofHG and RG-I, preferably at least 70% HG and less than 30% RG-I morepreferably at least 80% HG and less than 20% RG-I and most preferably atleast 90% HG and less than 10% RG-I. In certain embodiments, the pectinmay contain 0-10% of RG-II. In certain preferred embodiments, thestarting pectin is citrus pectin.

B. Structural Modification of Pectin

According to the invention, the term “modified pectin” herein refers toany changes to the structure of pectin that are brought about bychemical, physical or biological means or by some combination thereof.Non-limiting examples of changes to the pectin structure include (butare not limited to) deesterification, hydrolysis, oxidation and/orreduction of sugar moieties, functionalization of sugar moieties,conformational changes, and changes in molecular weight, linkage, statesof aggregation, and particle size.

In one embodiment, modified pectin has the structure of a pectic acidpolymer with some of the pectic side chains still present. In preferredembodiments, the modified pectin is a copolymer of homogalacturonic acidand rhamnogalacturonan I in which some of the galactose- andarabinose-containing sidechains are still attached. The modified pectinmay have a molecular weight of 1 to 500 kilodaltons (kD), preferably 10to 250 kD, more preferably 50-200 kD, 70-175 kD, 70-150 kD, or 80-150kD, or even 80 to 100 kD as measured by Gel Permeation Chromatography(GPC) with Multi Angle Laser Light Scattering (MALLS) detection.

Degree of esterification is another characteristic of modified pectins.In certain embodiments, the degree of esterification may be between 0and 80%, between 10 and 60%, between 0 and 50%, or between 20 and 60%,such as 20-45%, or 30-40% esterification.

Saccharide content is another characteristic of modified pectins. Incertain embodiments, the modified pectin is composed entirely of asingle type of saccharide subunit. In other embodiments, the modifiedpectin comprises at least two, preferably at least three, and mostpreferably at least four types of saccharide subunits. For example, themodified pectin may be composed entirely of galacturonic acid subunits.Alternatively, the modified pectin may comprise a combination ofgalacturonic acid and rhamnose subunits. In yet another example, themodified pectin may comprise a combination of galacturonic acid,rhamnose, and galactose subunits. In yet another example, the modifiedpectin may comprise a combination of galacturonic acid, rhamnose, andarabinose subunits. In still yet another example, the modified pectinmay comprise a combination of galacturonic acid, rhamnose, galactose,and arabinose subunits. In some embodiments, the galacturonic acidcontent of modified pectin is greater than 50%, preferably greater than60% and most preferably greater than 80%. In some embodiments, therhamnose content is less than 25%, preferably less than 15% and mostpreferably less than 10%; the galactose content is less than 50%,preferably less than 40% and most preferably less than 30%; and thearabinose content is less than 15%, preferably less than 10% and mostpreferably less than 5%. In certain embodiments, the modified pectin maycontain other uronic acids, xylose, ribose, lyxose, glucose, allose,altrose, idose, talose, gluose, mannose, fructose, psicose, sorbose ortalalose in addition to the saccharide units mentioned above.

Modified pectin suitable for use in the subject methods may also haveany of a variety of linkages or a combination thereof. By linkages it ismeant the sites at which the individual sugars in pectin are attached toone another. In some embodiments, the modified pectin comprises only asingle type of linkage. In certain preferred embodiments, the modifiedpectin comprises at least two types of linkages, and most preferably atleast 3 types of linkages. For example, the modified pectin may compriseonly alpha-1,4 linked galacturonic acid subunits. Alternatively, themodified pectin may comprise alpha-1,4-linked galacturonic acid subunitsand alpha-1,2-rhamnose subunits. In another example, the modified pectinmay be composed of alpha-1,4-linked galacturonic acid subunits andalpha-1,2-rhamnose subunits linked through the 4 position to arabinosesubunits. In another example, the modified pectin may comprisealpha-1,4-linked galacturonic acid subunits and alpha-1,2-rhamnosesubunits linked through the 4 position to arabinose subunits withadditional 3-linked arabinose subunits. In another example, the modifiedpectin may comprise alpha-1,4-linked galacturonic acid subunits andalpha-1,2-rhamnose subunits linked through the 4 position to arabinosesubunits with additional 5-linked arabinose units. In another example,the modified pectin may comprise alpha-1,4-linked galacturonic acidsubunits and alpha-1,2-rhamnose subunits linked through the 4 positionto arabinose subunits with additional 3-linked and 5-linked arabinosesubunits. In another example, the modified pectin may comprisealpha-1,4-linked galacturonic acid subunits and alpha-1,2-rhamnosesubunits linked through the 4 position to arabinose subunits withadditional 3-linked and 5-linked arabinose subunits with 3,5-linkedarabinose branch points. In another example, the modified pectin maycomprise alpha-1,4-linked galacturonic acid subunits andalpha-1,2-rhamnose subunits linked through the 4 position to galactosesubunits. In another example, the modified pectin may comprisealpha-1,4-linked galacturonic acid subunits and alpha-1,2-rhamnosesubunits linked through the 4 position to galactose subunits withadditional 3-linked galactose subunits. In another example, the modifiedpectin may comprise alpha-1,4-linked galacturonic acid subunits andalpha-1,2-rhamnose subunits linked through the 4 position to galactosesubunits with additional 4-linked galactose subunits. In anotherexample, the modified pectin may comprise alpha-1,4-linked galacturonicacid subunits and alpha-1,2-rhamnose subunits linked through the 4position to galactose subunits with additional 3-linked galactosesubunits with 3,6-linked branch points. In another example, the modifiedpectin may comprise alpha-1,4-linked galacturonic acid subunits andalpha-1,2-rhamnose subunits linked through the 4 position to galactosesubunits with additional 4-linked galactose subunits with 4,6-linkedbranch points. In certain embodiments, the side chains of the modifiedpectin may comprise uronic acids, galacaturonic acid, glucuronic acid,rhamnose, xylose, ribose, lyxose, glucose, allose, altrose, idose,talose, gluose, mannose, fructose, psicose, sorbose or talalose inaddition to the saccharide units described above.

Modified pectin according to the invention may have one, all or somesubcombination of the characteristics described above.

Modification by Chemical Methods

Modified pectin may be produced by chemical means. By chemical means itis meant to include any chemical reaction or process that disrupts orchanges chemical bonding of the pectin structure. Chemical bondingincludes any bonding which is readily known in the art. By way ofexample this may include covalent or ionic bonds. Chemical reactionsinclude any reaction known in the art and include those that eitherdirectly or indirectly alter chemical bonds. By way of example only,chemical bonding may be disrupted or formed by catalysis, hydrolysis,substitution, elimination, reduction, oxidation, and radical reactions.In certain embodiments, modified pectin according to the invention isproduced by a process that includes hydrolysis, which is preferablycatalyzed, e.g., by an acidic or basic reagent or both.

In one preferred embodiment, an aqueous solution of pectin is modifiedby the action of alkaline conditions. Under these conditions, theoriginal pectin structure may undergo several changes. Non-limitingexamples include de-esterification of the methylated galacturonic acidresidues, cleavage of the galacturonic acid backbone, cleavage of thebranch points, as well as conformational changes, for example fromrandom coil to rigid rod, or change in aggregation state.

In another preferred embodiment an aqueous pectin solution is modifiedby the action of acidic conditions. Under these conditions, thestructure of pectin may undergo several changes. These changes include,but are not limited to, de-esterification, hydrolysis of the backbone,branch points or side chains, conformational changes, for example fromrigid rod to random coil, and changes in aggregation state.

In yet another more preferred embodiment, an aqueous pectin solution istreated sequentially to alkaline conditions followed by acidicconditions or vice versa.

In another embodiment, pectin is modified by the addition of salts. Inone embodiment divalent salts such as calcium or magnesium are used toincrease the molecular weight of the modified pectin by ionic bonding.In another embodiment, high salt concentrations are used to causeconformational changes to the pectin structure by interruptingintramolecular ionic and hydrogen bonding.

In alternative embodiments, pectin is modified by oxidation. In onepreferred embodiment the C-6 hydroxyls of the galactose side chains areoxidized to carboxylic acids using methods known in the art, for exampleselective primary alcohol oxidation in aqueous solution using2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO).

In an alternative embodiment, pectin my also be modified by the use of areductant. For example, the methyl esters of the C-6 carboxyl group onthe galacturonic acid can be reduced to hydroxyl groups using sodiumborohydride.

Modification by Physical Methods

In another aspect of the invention pectin may be modified by physicalmeans. Physical means are meant to include non-chemical or biologicalmeans that alter the structure of pectin. Such physical means mightinclude, but are not limited to heat, cold, freeze/thaw, irradiation,shear, ultra-high shear, addition of cosolvents and other ionicdisrupting compounds, and filtration. In one embodiment, the particlesize polydisperisty of a colloidal solution of modified pectin isreduced by the use of microfiltration. Particularly, a solution ofmodified pectin may be filtered using tangential flow filtrationcomprising a particular membrane type and pore size. In anotherembodiment, a modified pectin solution may be combined with a secondsolvent such as ethanol to compress the particle size by reduction ofhydration prior to filtration.

The choice of filter pore size and membrane type, the amount ofcosolvent, the pH, and concentration depend on the degree ofpolydispersity desired. In preferred embodiments, the pore size utilizedis less than 1.0 μm, preferably less than 0.65 μm, more preferably lessthan 0.45 μm, and most preferably less than 0.22 μm. In other preferredembodiments, ethanol is the cosolvent and is present at a concentrationof less 0 to 40% w/w, more preferably 10 to 30% w/w, and most preferably15-25% w/w. In a preferred embodiment, a pectin solution or a chemicallymodified pectin solution is filtered at a concentration of 2.5 to 7.5mg/mL in 10 to 25% aqueous ethanol.

Modification by Biological Methods

In yet another aspect of the invention, pectin may be modified bybiological means. Biological means are meant to include by way ofexample, enzymatic degradation of pectin. For example, a pectinaseenzyme or cocktail of enzymes may be used to reduce the molecular weightof pectin. The choice of the enzyme(s) employed will depend upon thedesired outcome. In one embodiment, the enzymes may be selected tohydrolyze the polygalacturonic backbone. In another embodiment, theenzyme(s) may be selected to hydrolyze the side chains from thebackbone. In yet another embodiment, the enzyme or enzyme cocktail maybe selected to alter the degree of esterification.

Modification by Alternative Methods

Finally, the invention contemplates the preparation of modified pectinby synthetic and/or recombinant methods. Like the production ofrecombinant proteins, complex polysaccharides might be engineered via aseries of synthetic reactions or by recombinant methods.

In certain embodiments, the method of the invention includes anysubcombination of the foregoing acts.

C. Methods of Controlling Therapeutic Effectiveness

The therapeutic effectiveness of modified pectin can be controlled byseveral methods including controlling molecular weight and particle sizepolydispersity, the degree of esterification, the average molecularweight, the pH, and the charge density. In one embodiment, thetherapeutic effectiveness of a modified pectin is increased by reducingthe particle size polydispersity by means of microfiltration. In apreferred embodiment, the microfiltration is tangential flow filtration.The degree of therapeutic effectiveness can be increased or decreasedbased upon the filtration conditions utilized. In particular, thefiltration feed and filtration parameters can be adjusted to control thedegree of therapeutic activity of the modified pectin. In general, thetherapeutic activity of a modified pectin can be increased by one or acombination of the following parameters: adjusting the filtration feedto a lower pH prior to filtration, increasing the concentration of themodified pectin in the filtration feed, adding a cosolvent such asethanol or increasing the ratio of filtration membrane surface area toweight of filtered product. The invention contemplates that additionalparameters or other means can be employed to achieve the same result.For example, ultrasonication or high shear methods may also be employedto create smaller particle sizes.

Methods of Controlling Molecular Weight

The molecular weight of modified pectin may also be controlled, e.g.,increased or decreased. Increases in molecular weight may be broughtabout by formation of ionic interactions, hydrogen bonds, covalentbonds, or some combination thereof.

Decreases in molecular weight may be brought about by interruption ofionic interactions, hydrogen bonds, or covalent bonds, e.g., byacid-catalysed hydrolysis, base-catalyzed cleavage, or some combinationthereof. In one embodiment of the invention, the molecular weight ofpectin or modified pectin is reduced by hydrolysis of the covalentlinkages of main backbone, hydrolytic removal of the side chains, orsome combination thereof. In a specific example, the molecular weight isreduced by increasing the pH of an aqueous solution of pectin. The rateof the hydrolysis is controlled by pH and time. In one method, the pH ofthe solution is between 7-13. In a preferred method, the pH of thesolution is between 8.5-11.5, more preferably between 10-11.

In certain embodiments, the molecular weight is reduced by increasingthe temperature of either dry pectin, solutions of pectin, orsuspensions of pectin. The rate of decrease in molecular weight iscontrolled by temperature and time. In one method the temperature isbetween 50 and 200° C., more preferably between 80 and 150° C. and mostpreferably between 95 and 125° C. When decreasing the molecular weightusing heat, the type of vessel used may affect the rate of the reaction.For example, when heating a solution it may be optimal to conduct theheating in a closed vessel. This closed system will not allow loss ofvolume, but must be able to withstand the vapor pressure generated bythe reaction. Additionally, the presence of oxygen (either dissolved oratmospheric) may cause unwanted side reactions, such as oxidation. Thiscan be avoided by running the reaction in a closed vessel under vacuumor purged with nitrogen, or by other methods known in the art.

In certain embodiments, molecular weight may be affected by irradiation.High-energy radiation such as gamma irradiation is known to cause thescission or formation of new bonds through the formation of radicals.The degree of molecular weight modification is dependent on severalvariables such as intensity of the irradiation, exposure duration,dissolved or atmospheric oxygen, and density of material beingirradiated. However, in one method, an aqueous solution of pectin in aclosed vessel under vacuum is irradiated for at least 1 minute,preferably at least 10 minutes, more preferably at least 30 minutes andmost preferably at least 60 minutes and the intensity of the irradiationis at least 1 kilogray, preferably at least 5 kilogray, more preferably15 kilogray, and most preferably 25 or more kilogray.

In certain embodiments, the molecular weight may reduced by shear. Shearmay be produced by a number of methods which are known in the art, andmay include sonication and high shear mixing.

In certain embodiments, the molecular weight of a modified pectin may beincreased by the action of a chemical crosslinker. In one embodiment,the molecular weight of modified pectin is increased by crosslinking itwith a water soluble diamine, such as poly(ethyleneglycol)bisamine usinga carbodiimide intermediate.

Depending on the final product desired, the above methods for modulatingthe molecular weight may be performed individually or in somecombination.

Methods For Controlling Molecular Weight Polydispersity

In some instances it may be desirable to optimize the finalpolydispersity of the modified pectin. For example, modified pectins ofa particularly narrow molecular weight range or colloidal particle sizemay give optimal pharmaceutical performance.

The polydispersity of modified pectin can be controlled by themechanical removal of products having unwanted molecular weights. Incertain embodiments, the unwanted products are eliminated by one or morefractional precipitations. In certain preferred embodiments, unwantedproducts are removed by either one or a series of ultrafiltrations withdiffering nominal molecular weight cut-off membranes. Ultrafiltration isa well known process and typically takes place in an aqueous solvent.

The polydispersity can be reduced by using size exclusionchromatography. In this method, a modified pectin solution is passedthrough a size exclusion column which differentially separates moleculesbased on their molecular weight.

The polydispersity of the modified pectin can be controlled by itsmethod of manufacture. In one such method, the polydispersity of thepectin or modified pectin is decreased by exposing a solution orsuspension to ultrahigh shear. The action of shear, especially ultrahighshear, causes bonds of larger polymers to break preferentially at aboutthe middle of the polymer, leaving smaller polymers intact. This actionwill result in the preferential formation of smaller polymers ofapproximately the same size and thus a smaller polydispersity.

Methods for Controlling Monosaccharide Content

There are a wide variety of enzymes, including some produced by bacteriaand fungi, that selectively fragment particular segments of the pectinpolysaccharide. Various combinations of these enzymes may be employed toremove methyl esters, individual sugars, side chains, or sections of thebackbone as desired. Enzymes that can be used alone or in combination toalter monosaccharide composition include, for example,rhamnogalacturonase, rhamnogalacturonan lyase,rhamnogalacturonan-rhanmohydrolase,rhamnogalacturonan-galacturonohydrolase, endo and exo galacturonases,arabinases, pectin methyl esterases, and galactanases.

The monsaccharide content of modified pectin can be controlled by theconversion of one type of monosaccharide moiety into another. In oneembodiment of the invention, the galacturonic acid content of themodified pectin is reduced by the selective chemical reduction of thecarboxyl on C-6 of galacturonic acid to form galactose. Themonosaccharide content can also be affected by the source of the pectinstarting material, as discussed above.

Methods for Controlling Linkage

Linkages can be controlled by choice of the source of the startingpectin, the nature of the chemical process to produce the modifiedpectin, and enzymatically by selective cleavage with enzymes. In oneembodiment, acid hydrolysis is used to preferentially cleave the neutralside chains from the pectin backbone.

D. Methods of Purification

Typically, the processes of making modified pectin result in a mixtureof modified pectin and a number of impurities such as low molecularweight by-products, salts, co-solvents and inactive modified pectin. Incertain embodiments of the invention, these impurities are acceptableand modified pectin is not further purified or isolated. In other morepreferred embodiments, the impurities are either reduced or removedentirely from the modified pectin composition.

According to the invention, impurities can be removed by methods knownin the art. By way of example, these methods include filtration,microfiltration, ultrafiltration, chromatography, centrifugation,extraction, drying, precipitation and dialysis.

Impurities may be separated from modified pectin by screen filtration,microfiltration, ultrafiltration, or a combination thereof. For example,microfiltration of an aqueous modified pectin solution can be used toremove insoluble materials from the solution, while salts and lowmolecular weight by-products can be removed by using ultrafiltration.The choice of the membrane and its nominal molecular weight cut-offdepends on the molecular size of the impurities which are to be removedand the molecular weight of the modified pectin product desired. Incertain embodiments, a solution of modified pectin is purified by screenfiltration using a 5 μm filter screen, by microfiltration through a 0.2μm filter, by ultrafiltration through a 30 kilodalton ultra-filter, orany combination of these.

Modified pectin may also be purified by chromatography. The type ofchromatography is selected based on the nature of the impurities to beremoved; methods for choosing a suitable type of chromatography are wellknown in the art. Exemplary types of chromatography include sizeexclusion, ion exchange, and affinity chromatography.

Solvents, such as ethanol, acetone, water, etc., can be removed by anymeans known in the art, such as by drying or evaporation. In certainembodiments, solvents may be removed by lyophilization or vacuumdistillation.

E. Methods of Formulating Modified Pectin Compositions

Any or all of the above methods may be combined to formulate modifiedpectin compositions suitable for therapeutic uses.

In certain embodiments, preparing modified pectin comprises partiallydepolymerizing a pectin polymer by disrupting the rhamnogalacturonanbackbone, deesterifying galacturonic acid moieties in the backbone, andbreaking down side chains of neutral sugars. In certain embodiments, themethod preferably generates a substantially ethanol-free productsuitable for parenteral administration.

In one exemplary method of the invention, preparing modified pectincompositions comprises:

dissolving the pectin in water at a concentration of 0.1 to 40 mg/mL,preferably 1 to 20 mg/mL, more preferably 5 to 15 mg/mL and mostpreferably about 10 mg/mL;

depolymerizing and de-esterifying the pectin by digestion, e.g., at analkaline pH of 7 to 13, preferably 9 to 12, more preferably 10 to 11,and most preferably about 10.7, for a duration of less than about 24hours, preferably less than 12 hours, more preferably less than 1 hour,and most preferably about 10-30 minutes;

further digesting the pectin at an acidic pH from 1 to 6.9, preferably 2to 5, more preferably from 2.5 to 3.5, and most preferably about 3, fora duration of several days, preferably about 24 hours, preferably lessthan 12 hours, more preferably less than 1 hour, and most preferablyabout 5-15 minutes;

neutralizing or adjusting the solution to a pH from 4 to 8, preferablyfrom 5 to 8, more preferably from 6 to 8, and most preferably from 6 to7;

precipitating and washing the modified pectin with ethanol;

dissolving the washed precipitate in water;

filtering the solution through a filter, e.g. a 0.2 μm filter; and

concentrating and diafiltering the solution of modified pectin byultrafiltrationusing, e.g., a 30 kD—molecular weight cut-off membrane.

In certain embodiments, the method further includes adjusting thesolution to isoosmolality and/or clarifying the solution. In certainembodiments, the method further includes fractionating the modifiedpectin molecules by size. Sizing can include fractionation of differentparticle sizes, molecular weight, particle or molecular shape. Themethod may further include sterile filtration, addition of sterilefilling, removal of endotoxins, lyophilization, or any combination ofthese steps. Preferably, all of these additional steps are performed.

In certain embodiments of the above described method, dissolving thepectin comprises providing a slurry of pectin in a water-miscibleorganic solvent and combining the slurry with water to dissolve thepectin. The use of a slurry of pectin in a water-miscible organicsolvent accelerates the dissolution and/or prevents the aerialdispersion of pectin particles that occurs when dry pectin is addeddirectly to an aqueous solvent. The organic solvent preferably is orcontains an alcohol, and more preferably the solvent is ethanol.

The alkaline pH as described above may be obtained by addition of analkaline salt or a solution thereof, or in an alternative embodiment thewater used to dissolve the pectin may already contain a dissolvedalkaline salt and have a pH in the desired range. Suitable alkalinesalts include but are not limited to sodium hydroxide, sodium carbonate,potassium hydroxide, and potassium carbonate. Preferably, sodiumhydroxide is employed.

The acidic pH as described above may be achieved by addition of an acid,preferably a mineral acid or solution thereof. Examples of suitablemineral acids include but are not limited to hydrochloric acid, sulfuricacid, and acid salts such as sodium bisulfate and monobasic sodiumphosphate.

The neutralization process as described above involves the addition ofan alkaline salt or a solution of an alkaline salt, as described above.Optionally, a buffer such as a phosphate or carboxylate salt may beintroduced as an aid to the neutralization process, for example tominimize local extremes in pH.

In certain embodiments, filtration of the dissolved precipitate asdescribed above is carried out using tangential flow filtration. Theeffect of the filtration on the modified pectin can be influenced by themake-up of the feed solution (such as modified pectin concentration,cosolvent concentration, pH, conductivity, and temperature) and thefiltration operating parameters utilized (transmembrane flow rate,filter surface area, and retentate flow rate). In one preferredembodiment, the modified pectin is filtered using a 0.2 μm tangentialflow filter.

In preferred embodiments, preparing modified pectin comprisesultrafiltration of the solution of modified pectin (e.g., using a 30 kDmembrane) as described above.

IV. Delivery of Modified Pectin

In certain embodiments, the compositions are suitable for parenteraladministration to a mammal, most preferably by injection or intravenousinfusion, and in some embodiments the compositions may comprise one ormore pharmaceutically acceptable excipients. Suitable excipients includepharmaceutically acceptable buffers, stabilizers, local anesthetics, andthe like. The composition may be adapted for direct injection orintravenous infusion, or for addition to an intravenous drip solutionfor gradual infusion, through appropriate use of excipients andpackaging and delivery means well known in the art.

In another aspect, the invention provides a pharmaceutical package,comprising a vial or ampoule containing a modified pectin according tothe invention in the form of a reconstitutable powder or a solutionsuitable for injection or infusion, together with instructions foradministering the composition to a patient in need thereof. Instructionsinclude but are not limited to written and/or pictorial descriptions of:the active ingredient, directions for diluting the composition to aconcentration suitable for administration, suitable indications,suitable dosage regimens, contraindications, drug interactions, and anyadverse side-effects noted in the course of clinical trials.

In an alternative embodiment of the above aspect of the invention, thepharmaceutical package may comprise a plastic bag containing from 100 mlto 2 L of a pharmaceutical composition of the invention, in the form ofa solution suitable for intravenous administration, together withinstructions as described above.

In alternative embodiments, a pharmaceutical composition of theinvention may be in a form adapted for oral dosage, such as for examplea syrup or palatable solution; a form adapted for topical application,such as for example a cream or ointment; or a form adapted foradministration by inhalation, such as for example a microcrystallinepowder or a solution suitable for nebulization. Methods and means forformulating pharmaceutical ingredients for alternative routes ofadministration are well-known in the art, and it is to be expected thatthose skilled in the relevant arts can adapt these known methods to themodified pectins of the invention.

The present invention provides pharmaceutically acceptable compositionscomprising a therapeutically effective amount of one or more of themodified pectins of the invention, formulated together with one or morepharmaceutically acceptable excipients. The pharmaceutical compositionsof the present invention may be formulated for administration in solidor liquid form, including forms adapted for oral administration, forexample, aqueous or non-aqueous solutions or suspensions, tablets,powders, and granules; administration by inhalation, for example,aerosols, solutions for nebulization, or dry powders; parenteraladministration, for example sterile solutions or suspensions; topicalapplication, for example lotions, creams, ointments or sprays;ophthalmic administration; or intravaginal or intrarectaladministration, for example pessaries, suppositories, creams or foams.Preferably, the pharmaceutical preparation is adapted for parenteraladministration, more preferably it is a non-pyrogenic solution adaptedfor intravenous administration.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention may optionally be scored orprepared with coatings and shells, such as enteric coatings and othercoatings well known in the pharmaceutical-formulating art. They may alsobe formulated so as to provide slow or controlled release of themodified therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile, otherpolymer matrices, liposomes and/or microspheres. They may be sterilizedby, for example, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions that can be dissolved in sterile water, or some othersterile injectable medium immediately before use. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally in a delayedmanner. Examples of embedding compositions that can be used includepolymeric substances and waxes. The modified pectin can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Liquid dosage forms for oral administration of the modified pectins ofthe invention include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the modified pectin, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, solubilizing agents and emulsifiers.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

In dry powder formulations adapted for inhalation, the particle size ofthe particulate medicament should be such as to permit inhalation ofsubstantially all of the medicament into the lungs upon administrationof the aerosol formulation and will thus desirably be less than 20microns, preferably in the range 1 to 10 microns, more preferably 1 to 5microns. The particle size of the medicament may be reduced byconventional means, for example by milling or micronisation. The aerosolformulation preferably contains 0.5-30% w/w of modified pectin relativeto the total weight of the formulation.

The propellant may optionally contain an adjuvant having a higherpolarity and/or a higher boiling point than the propellant. Polaradjuvants which may be used include (e.g. C₂₋₆) aliphatic alcohols andpolyols such as ethanol, isopropanol and propylene glycol, preferablyethanol. In general only small quantities of polar adjuvants (e.g.0.05-3.0% w/w) may be required to improve the stability of thedispersion. However, the formulations of the invention are preferablysubstantially free of polar adjuvants, especially ethanol. Suitablepropellants include trichlorofluoromethane (propellant 11),dichlorodifluoromethane (propellant 12), dichlorotetrafluoroethane(propellant 114), tetrafluoroethane (propellant 134a) and1,1-difluoroethane (propellant 152a), saturated hydrocarbons such aspropane, n-butane, isobutane, pentane and isopentane, and alkyl etherssuch as dimethyl ether. In general, up to 50% w/w of the propellant maycomprise a volatile adjuvant, for example 1 to 30% w/w of a volatilesaturated C1-C6 hydrocarbon.

The aerosol formulations according to the invention may optionallycomprise one or more surfactants that are physiologically acceptableupon administration by inhalation.

For administration by inhalation, the drug is suitably inhaled from anebulizer, from a pressurized metered dose inhaler or as a dry powderfrom a dry powder inhaler optionally using gelatin, plastic or othercapsules, cartridges, blister packs and/or strips.

Administration of medicament may be indicated for the treatment of mild,moderate or severe acute or chronic symptoms or for prophylactictreatment. It will be appreciated that the precise dose administeredwill depend on the age and condition of the patient, the particularparticulate medicament used and the frequency of administration and willultimately be at the discretion of the attendant physician. Typically,administration will range from one or to four or more times daily.

For use in dry powder inhalers, the active ingredient can be modified byspray drying or compression to form a powder with suitable flowproperties. More commonly a diluent or carrier is added which isgenerally non-toxic and inert to the medicament. Examples of suchcarriers are polysaccharides e.g. starch and cellulose, dextran,lactose, glucose, mannitol, and trehalose. The carrier can be furthermodified by the addition of surface modifiers, pretreatment to form lowrugosity particles, addition of glidants, and flavor masking ormodifying agents.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise a modified pectin of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or non-aqueous solutions, or sterile powders which maybe reconstituted into sterile injectable solutions or dispersions justprior to use, which may contain antioxidants, buffers, bacteriostats,solutes which render the formulation isotonic with the blood of theintended recipient or suspending or thickening agents.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions.

Examples of pharmaceutically acceptable antioxidants include but are notlimited to ascorbic acid, cysteine hydrochloride, sodium metabisulfite,sodium sulfite, ascorbyl palmitate, butylated hydroxyanisole (BHA),butylated hydroxytoluene (BHT), propyl gallate, alpha-tocopherol, andchelating agents such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The modified pectin maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants that may berequired.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof. Ophthalmic formulations, eyeointments, powders, solutions and the like, are also contemplated asbeing within the scope of this invention.

Formulations of the present invention which are suitable for vaginaladministration include pessaries, tampons, creams, gels, pastes, foamsor spray formulations, containing such carriers as are known in the artto be appropriate. Such formulations may be prepared, for example, bymixing one or more modified pectins of the invention with one or moresuitable nonirritating excipients comprising, for example, cocoa butter,polyethylene glycol, or a suppository wax, which is solid at roomtemperature but liquid at body temperature and, therefore, will melt inthe rectum or vaginal cavity and release the active modified pectin.

V. Uses of Modified Pectin

In another aspect, the invention provides a method of inhibiting a cellproliferation process in a patient, which comprises administering apharmaceutical composition of the invention to the patient, therebyinhibiting the cell proliferation process. Cell proliferative processesinclude but are not limited to angiogenesis and cell proliferativedisorders such as psoriasis, endometriosis, benign hyperplasias, andvarious types of cancer, including renal cell cancer, Kaposi's sarcoma,chronic leukemia, chronic lymphocytic leukemia, lymphoma, mesothelioma,breast cancer, sarcoma, myeloma, ovarian carcinoma, rectal cancer,throat cancer, melanoma, colon cancer, bladder cancer, mastocytoma, lungcancer, liver cancer, mammary adenocarcinoma, pharyngeal squamous cellcarcinoma, prostate cancer, pancreatic cancer, gastrointestinal cancer,and stomach cancer.

In yet another aspect, the invention provides a method of enhancing theeffects of conventional cancer treatments including chemotherapy,radiation therapy, surgery, and combinations thereof. Additionally, theinvention provides treatments of other hyperproliferative disorders,diseases associated with corneal neovascularization, diseases associatedwith chronic inflammation, and autoimmune diseases. Further disclosureof related compositions and use are disclosed in U.S. Pat. No. 6,680,306and U.S. patent application Ser. Nos. 08/024,487, 10/299,478,10/176,022, and 60/461,006 the disclosures of which are incorporatedherein by reference.

The invention also provides a method for conducting a medical assistancereimbursement program, the method comprising: (a) providing areimbursement program that permits, for prescription of a composition,solution, or depolymerized pectin of the invention for treating a canceror other disorder associated with cell proliferation, at least partialreimbursement to a healthcare provider or patient, or payment to a drugdistributor; (b) processing one or more claims for reimbursement of thecost of a prescription of the composition, solution, or depolymerizedpectin for treating a cancer; and (c) reimbursing the healthcareprovider or patient, or paying a drug distributor, at least a portion ofthe cost of said prescription.

EXAMPLES

By way of example, and not limitation, the following examples areprovided. Modified pectin materials were prepared as described herein,and the therapeutic affects of modified pectin were evaluated using twoin vitro models, Cell Apoptosis Model, described in Example 4, and DNASynthesis Inhibition Model, described in Example 5.

Example 1 Preparation of Modified Pectin

Citrus fruit pectin (800 g) was added (at a rate of about 15 g/min) withvigorous stirring to water (89 liters). Following addition of thepectin, the mixture was stirred for approximately 1 hour until thepectin appeared dissolved. The solution was then rapidly adjusted to pH10.7 by the addition of ˜10 N NaOH solution, and stirred at about 27°C.) for 20 minutes, while maintaining pH 10.7 using ˜10 N NaOH.Following this the solution pH was adjusted to pH 3.0 by gradualaddition of 3 M HCl and maintained for 10 minutes. The pH was thenadjusted to 6.3 using 10 M and 1M NaOH and maintained for 10 minutes.

The resulting solution was then transferred into a 70% ethanol solutionto precipitate the modified pectin. The precipitate was then isolated byscreen filtration and washed with a 70% ethanol solution.

The precipitate was then dissolved in water, adjusted to 5 mg/mLmodified pectin, 15% w/w ethanol and pH 6.5. The resulting solution wasthen filtered through a 0.2 μm tangential flow filter. This was followedby ultrafiltration and diafiltration through a 30 kD membrane toconcentrate the modified pectin and reduce low molecular weightimpurities including ethanol. The solution was then diluted with waterto a final modified pectin concentration of 5 mg/mL.

This final solution was then filtered through 0.2 μm filters and filledinto sterile glass vials. The modified pectin composition obtained bythis process exhibited an ethanol content of less than 1%, an averagemolecular weight of approximately 90 kD as judged by a multiangle,light-scattering chromatography method, a modified pectin concentrationof 5±0.5 mg/mL as determined by a size exclusion chromatography method.The composition was tested by the cancer Cell Apoptosis and DNASynthesis Inhibition models described in Example 4 and 5, respectively,and found to be highly effective in both, with an average IC₅₀=50 μg/mL.

Example 2 Effect of Microfiltration Conditions on TherapeuticEffectiveness

The effect of varying microfiltration conditions on the control oftherapeutic effectiveness was demonstrated by varying differentfiltration parameters and testing the modified pectin for activity inthe cancer Cell Apoptosis model. Modified pectin was fractionated bysize using microfiltration. FIG. 1 demonstrates that the samplesprepared using different microfiltration pore sizes have differenttherapeutic effectiveness. In the range used in this experiment, thelargest pore size (0.7 μm) had the least therapeutic effect while thesmallest pore size (0.1 μm) had the most therapeutic effect, asdetermined by the Cell Apoptosis model.

The pH of a sample that is filtered by the microfiltration procedure(“microfiltration feed”) also has an effect on the therapeuticeffectiveness of the prepared sample. FIG. 2 demonstrates therelationship between microfiltration feed pH and therapeuticeffectiveness. In the range used in this experiment, the samplesprepared from lower feed pH demonstrated higher therapeutic effect, asdetermined by the Cell Apoptosis model.

Another factor that influences the therapeutic effectiveness of modifiedpectin is the ethanol content of the microfiltration feed. FIG. 3demonstrates the effect of microfiltration feed ethanol content ontherapeutic effectiveness. In the range used in this experiment, thesamples processed with the higher ethanol concentrations demonstratedhigher therapeutic effect, as determined by the Cell Apoptosis model.

For the three parameters tested, the modified pectin was prepared asdescribed in Example 1, except the tangential flow filtration feedsolution was modified appropriately.

Example 3 Modified Pectin Produced Via Physical Methods Only

As disclosed above, pectin can be modified by physical methods alone.This was demonstrated by preparing a pectin solution and processing bytangential flow filtration using filter feed conditions provided in theExample 1. The resulting material was tested for therapeutic activity inthe cancer cell apoptosis model and it was found to have an IC₅₀ of 46μg/mL whereas the unfiltered pectin feed solution had no detectabletherapeutic activity.

Example 4 Cell Apoptosis Therapeutic Model

An experimental model to assess a potential anti-cancer agent is CellApoptosis Therapeutic Model. In this model, cancer cell apoptosis wasdemonstrated using an in vitro bioactivity assay. The test isqualitative and is based on the observation that modified pectininhibits the proliferation of cancer cells such as B16-F10 mousemelanoma cells and induces cancer cell apoptosis. This effect can bemeasured by using a mitochondrial enzyme indicator such as AlamarBlue™(BioSource), which is formulated to quantitatively measure theproliferation or viability of a variety of human or animal cells in thepresence of adding toxic compounds. It consists of anoxidation-reduction (REDOX) indicator that yields a colorimetric changeand a fluorescent signal in a response to the cell innate metabolicactivity change, thereby providing an indirect measure of viable cellnumber. By testing several concentrations of the modified pectin, it ispossible to calculate an IC₅₀ for the compound measured in μg/milliliterand the smaller the IC₅₀ the greater the therapeutic effect.

B16F10 cells were seeded into the wells of 96-well plates in growthmedia. After cells attached to the plates, 0, 5, 50, or 150 μl/ml ofsterile 4-chlorodiazepam and 100 μl/ml of a sample to be tested wereapplied to the cells. The cells were incubated to allow viable cells toproliferate, and the media was replaced with fresh media supplementedwith 10% AlamarBlue™. The cells were further incubated, then the stateof oxidation of AlamarBlue™ was determined by spectrophotometry andadjusted for blanks. The more reduced AlamarBlue™ was, the higher theproportion of the cells that underwent apoptosis.

Example 5 DNA Synthesis Inhibition Therapeutic Model

The ability of the compound of the present invention to inhibit DNAsynthesis was assessed by the BrdU assay. The BrdU assay measures denovo DNA synthesis as an indicator of cell proliferation. BrdU(5-bromo-2′-deoxy-uridine) is a nucleotide analogue that substitutes forthymidine during DNA synthesis in proliferating cells. Incorporated BrdUis proportional to the amount of newly synthesized DNA and can bedetected by anti-BrdU monoclonal antibodies in ELISA, flow cytometry, orimmunohistochemistry.

To compare the effects of modified citrus pectin (MCP) of the currentinvention to MCP generated by previously disclosed methods on cellproliferation, BrdU incorporation in B16-F 10 mouse melanoma cells(ATCC, Manassas, Va.) was measured by Cell Proliferation ELISA, BrdU(Roche Applied Science, Indianapolis, Ind.). B16-F10 cells were grown ina 50:50 v/v media composed of EMEM and Leibovitz L-15 (both from CambrexBio Science, Walkersville, Md.) supplemented with 10% fetal bovine serum(JRH Bioscience, Lenexa Kans.), glutamine (JRH Bioscience), andpencillin-streptomycin (Invitrogen, Carlsbad, Calif.). Cells were seededat 3,000/well in 96-well white-walled, clear bottom microtiter plates(Corning Life Sciences, Acton, Mass.) and grown for 24 h. Thesupernatant was removed and replaced with the MCP's dissolved in thesupplemented 50:50 media. BrdU was added immediately following theagents. After 24 h agent and BrdU exposure, the cells were fixed andBrdU was detected with peroxidase-conjugated anti-BrdU. The luminescentsignal was developed with luminol and hydrogen peroxide and measuredwith a HT Synergy plate reader (Bio-Tek Instruments, Winooski, Vt.).

Example 6 Demonstration of Improved Therapeutic Activity for ModifiedPectin Prepared by Using the Methods of the Current Invention VersusPrevious Methods

The improved therapeutic activity of modified pectin using the methodsdisclosed in the current invention versus previously described methodswas demonstrated by preparing material according to the method describedin U.S. Application Publication No. U.S. 2002/0107222A1 and comparing itagainst material prepared using the methods described herein.

A first sample was prepared by the previous method described in U.S.2002/0107222A1. Briefly, pectin was dissolved in water at aconcentration of about 5 mg/mL. Using a 3 N sodium hydroxide (NaOH)solution, the pH was adjusted to 10 and maintained for 34 minutes. Thiswas followed by acidification to pH 3 using 3 N NaOH. The solution wasthen incubated at room temperature (˜17° C.) with stirring for 10 hours.The pH was then adjusted to pH 6.3 with 1 N NaOH. The resulting solutionwas then poured into a stirring solution of 70% w/w ethanol (2 parts 70%ethanol to 1 part solution) and left to precipitate for ˜16 hours. Theresulting precipitate was collected using vacuum filtration followed bywashing with 70% ethanol. The collected precipitate was then dried byseveral suspensions in acetone followed by filtration. The resultingpowder was then placed under vacuum for 10 hours to remove the residualsolvent. A 5 mg/mL solution was prepared and labeled as JR017047-2.

A second sample was prepared using the methods described in Example 1,except the material collected and washed by screen filtration was thendried using several acetone suspensions and filtrations. The resultingpowder was then dried under vacuum to remove the residual solvent. A 5mg/mL solution was prepared and labeled JR017053-2.

A third sample was prepared using the methods described in Example 1 andlabeled KB027002-9. Additionally, as a fourth sample, a sample of thetangential flow filtration feed solution was taken and labeledKB027002-8.

A fifth and six samples were prepared using the methods outlined inExample 1 and labeled 6612 and BB023013-10.

All of the above samples were tested by the Cell Apoptosis therapeuticmodel. In addition, all samples except the third and fourth samples weretested by the DNA Synthesis Inhibition therapeutic model.

Table 4 outlines the Cell Apoptosis therapeutic testing results from thesix samples outlined above. As indicated, the materials produced usingmethods of the current invention (third, fifth, and sixth sample,labeled KB027002-9, BB023013-10, and 6612 respectively) exhibitedgreater therapeutic effectiveness compared to the formerly disclosedmethods. Additionally, the data indicates this greater effectiveness isdirectly related to the content of the current invention, namely theinclusion of the additional filtration steps to control particle sizeand molecular weight polydispersity.

TABLE 4 Therapeutic effect of modified pectin samples prepared byprevious and new procedures Cell Apoptosis Model Sample ID IC50 μg/mLJR017047-2 289  JR017053-2 Inactive KB027002-9 43 KB027002-8 InactiveBB023013-10 34 6612 67

FIG. 4 show the DNA synthesis inhibition testing results for the fourcompounds tested (as outlined above). Like the Cell Apoptosis model,this data clearly shows the improved therapeutic effectiveness of themodified pectin that can be achieved as a result of using the methods ofthe current invention. When the cells that have been treated by the 6612sample was washed and re-seeded in a medium free of modified pectin, theDNA synthesis level returned to that of the control, indicating that theinhibition of DNA synthesis by the presence of 6612 is reversible.

All references cited within the specification are incorporated byreference in their entireties. The foregoing discussion and descriptionis illustrative of specific embodiments, but is not meant to be alimitation upon the practice thereof. It is the following claims,including all equivalents, which define the scope of the invention.

We claim:
 1. A process for manufacturing a biologically active filteredpectin, comprising: (a) passing an aqueous solution comprising 1-20mg/mL modified or unmodified pectin and 7.5%-25% w/w ethanol through atangential flow filter with a pore size of less than 1.0 μm to provide apermeate and a retentate, wherein said permeate comprises biologicallyactive filtered pectin having an average molecular weight from 10 kDa to250 kDa and an IC₅₀ less than 200 μg/mL in a cancer cell apoptosismodel; and (b) collecting said biologically active filtered pectin. 2.The process of claim 1, wherein the pore size is about 0.2 μm.
 3. Theprocess of claim 1, wherein the solution has a pectin concentration of 1to 10 mg/mL.
 4. The process of claim 1, wherein the solution has a pH inthe range of 2.5 to 7.5.
 5. A process for manufacturing a partiallyde-esterified biologically active pectin, comprising: (a) treating afirst aqueous solution comprising 1-20 mg/ml modified or unmodifiedpectin with acid, base, or both to partially de-esterify the pectin, (b)neutralizing the first aqueous solution to form a second aqueoussolution comprising partially de-esterified pectin, (c) passing thesecond aqueous solution comprising partially de-esterified pectin and7.5%-25% w/w ethanol through a tangential flow filter with a pore sizeof less than 1.0 μm to provide a permeate and a retentate, wherein saidpermeate comprises filtered partially de-esterified biologically activepectin having an average molecular weight from 10 kDa to 250 kDa and anIC₅₀ less than 200 μg/mL in a cancer cell apoptosis model, and whereinthe degree of esterification in the resulting partially de-esterifiedbiologically active pectin is between 10% and 60%, and (d) collectingsaid filtered partially de-esterified biologically active pectin.
 6. Theprocess of claim 5, wherein the pore size is about 0.2 μm.
 7. Theprocess of claim 5, wherein the first aqueous solution comprises 1 to 10mg/ml modified or unmodified pectin.
 8. The process of claim 5, whereinthe first aqueous solution has a pH in the range of 2.5 to 7.5.
 9. Aprocess for manufacturing a biologically active filtered pectin,comprising: (a) passing an aqueous solution comprising 1-20 mg/mLmodified or unmodified pectin and 7.5%-25% w/w ethanol through atangential flow filter with a pore size of 0.1 μm to 1.0 μm to provide apermeate and a retentate, wherein said permeate comprises biologicallyactive filtered pectin having an average molecular weight from 10 kDa to250 kDa and an IC₅₀ less than 200 μg/mL in a cancer cell apoptosismodel; and (b) collecting said biologically active filtered pectin. 10.The process of claim 9, wherein the pore size is 0.1 μm to 0.7 μm. 11.The process of claim 9, wherein the pore size is about 0.2 μm.
 12. Theprocess of claim 9, wherein the solution has a pectin concentration of 1to 10 mg/mL.
 13. The process of claim 9, wherein the solution has a pHin the range of 2.5 to 7.5.
 14. A process for manufacturing a partiallyde-esterified biologically active filtered pectin, comprising: (a)treating a first aqueous solution comprising 1-20 mg/ml modified orunmodified pectin with acid, base, or both to partially de-esterify thepectin, (b) neutralizing the first aqueous solution to form a secondaqueous solution comprising partially de-esterified pectin, (c) passingthe second aqueous solution comprising partially de-esterified pectinand 7.5%-25% w/w ethanol through a tangential flow filter with a poresize of 0.1 μm to 1.0 μm to provide a permeate and a retentate, whereinsaid permeate comprises filtered partially de-esterified biologicallyactive filtered pectin having an average molecular weight from 10 kDa to250 kDa and an IC₅₀ less than 200 μg/mL in a cancer cell apoptosismodel, and wherein the degree of esterification in the resultingpartially de-esterified pectin is between 10% and 60%, and (d)collecting said partially de-esterified biologically active filteredpectin.
 15. The process of claim 14, wherein the pore size is about 0.2μm.
 16. The process of claim 14, wherein the first aqueous solutioncomprises 1 to 10 mg/ml modified or unmodified pectin.
 17. The processof claim 14, wherein the first aqueous solution has a pH in the range of2.5 to 7.5.
 18. The process of claim 1, wherein the average molecularweight of the biologically active filtered pectin is selected from thegroup consisting of 50 kDa-200 kDa, 80 kDa-150 kDa, 70 kDa-150 kDa and80 kDa-100 kDa.
 19. The process of claim 5, wherein the averagemolecular weight of the biologically active filtered pectin is selectedfrom the group consisting of 50 kDa-200 kDa, 80 kDa-150 kDa, 70 kDa-150kDa and 80 kDa-100 kDa.
 20. The process of claim 9, wherein the averagemolecular weight of the biologically active filtered pectin is selectedfrom the group consisting of 50 kDa-200 kDa, 80 kDa-150 kDa, 70 kDa-150kDa and 80 kDa-100 kDa.
 21. The process of claim 14, wherein the averagemolecular weight of the biologically active filtered pectin is selectedfrom the group consisting of 50 kDa-200 kDa, 80 kDa-150 kDa, 70 kDa-150kDa and 80 kDa-100 kDa.
 22. The process of claim 1, wherein thebiologically active filtered pectin inhibits cancer cell proliferationin a cancer cell apoptosis model with an IC₅₀ less than 100 μg/mL. 23.The process of claim 5, wherein the biologically active filtered pectininhibits cancer cell proliferation in a cancer cell apoptosis model withan IC₅₀ less than 100 μg/mL.
 24. The process of claim 9, wherein thebiologically active filtered pectin inhibits cancer cell proliferationin a cancer cell apoptosis model with an IC₅₀ less than 100 μg/mL. 25.The process of claim 14, wherein the biologically active filtered pectininhibits cancer cell proliferation in a cancer cell apoptosis model withan IC₅₀ less than 100 μg/mL.
 26. The process of claim 1, wherein thesolution comprises 10%-20% w/w ethanol or 15%-25% w/w ethanol.
 27. Theprocess of claim 5, wherein the second aqueous solution comprises10%-20% w/w ethanol or 15%-25% w/w ethanol.
 28. The process of claim 9,wherein the solution comprises 10%-20% w/w ethanol or 15%-25% w/wethanol.
 29. The process of claim 14, wherein the second aqueoussolution comprises 10%-20% w/w ethanol or 15%-25% w/w ethanol.